Sialidase (neuraminidase, EC 3.2.1.18) catalyses the hydrolysis of terminal sialic acid residues of glyconjugates. Sialidase has been well studied in viruses and bacteria where it destroys the sialic acid-containing receptors at the surface of host cells, and mobilizes bacterial nutrients. In mammals, three types of sialidases, lysosomal, plasma membrane and cytosolic, have been described. For lysosomal sialidase in humans, the primary genetic deficiency results in an autosomal recessive disease, sialidosis, associated with tissue accumulation and urinary excretion of sialylated oligosaccharides and glycolipids. Sialidosis includes two main clinical variants: late-onset, sialidosis type I, characterized by bilateral macular cherry-red spots and myoclonus, and infantile-onset, sialidosis type II, characterized by skeletal dysplasia, mental retardation and hepatosplenomegaly. We report the identification of human lysosomal sialidase cDNA, its cloning, sequencing and expression. Examination of six sialidosis patients revealed three mutations, one frameshift insertion and two missense. We mapped the lysosomal sialidase gene to human chromosome 6 (6p21.3), which is consistent with the previous chromosomal assignment of this gene in proximity to the HLA locus.

Galactosialidosis is an inherited lysosomal storage disease caused by the combined deficiency of lysosomal sialidase and β-galactosidase secondary to the deficiency of cathepsin A/protective protein, which is associated with sialidase and β-galactosidase in a high-molecular weight (1.27 MDa) complex. Clinical phenotypes of patients as well as the composition of compounds which are stored in patient's tissues implicate sialidase deficiency as the underlying pathogenic defect. The recent cloning and sequencing of lysosomal sialidase [Pshezhetsky, Richard, Michaud, Igdoura, Wang, Elsliger, Qu, Leclerc, Gravel, Dallaire and Potier (1997), Nature Genet. 15, 316-320] allowed us to study the molecular mechanism of sialidase deficiency in galactosialidosis. By Western blotting, using antibodies against the recombinant human enzyme, and by NH2-terminal sequencing, we showed that sialidase is synthesized as a 45.5 kDa precursor and after the cleavage of the 47-amino acid signal peptide and glycosylation becomes a 48.3 kDa mature active enzyme present in the 1.27 kDa complex. Transgenic expression of sialidase in cultured skin fibroblasts from normal controls and from galactosialidosis patients, followed by immunofluorescent and immunoelectron microscopy showed that in both normal and affected cells the expressed sialidase was localized on lysosomal and plasma membranes, but the amount of sialidase found in galactosialidosis cells was ~ 5-fold reduced. Metabolic labelling studies demonstrated that the 48.3 kDa mature active form of sialidase was stable in normal fibroblasts (half-life ~ 2.7 h), whereas in galactosialidosis fibroblasts the enzyme was rapidly converted (half-life ~ 30 min) into 38.7 and 24 kDa catalytically inactive forms. Altogether our data provide evidence that the molecular mechanism of sialidase deficiency in galactosialidosis is associated with abnormal proteolytic cleavage and fast degradation.

Lysosomal neuraminidase (sialidase; EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23), together with a carboxypeptidase, the so-called 'protective protein', were co-purified from the human placenta by affinity chromatography on a concanavalin A-Sepharose column followed by a thiogalactoside-agarose affinity column for beta-galactosidase. Analysis of the purified material by gel-filtration h.p.l.c. revealed three distinct molecular forms, all with high beta-galactosidase specific activity, but only the largest one expressed neuraminidase activity. Rechromatography of each individual species separately indicated that all three are in fact part of an equilibrium system (the neuraminidase-beta-galactosidase-carboxypeptidase complex or NGC-complex) and that these species undergo slow conversion into one another through dissociation and association of protomeric components. Each species was sufficiently stable for the determination of their hydrodynamic properties by gel-filtration h.p.l.c. and sedimentation velocity. The largest species had an apparent sedimentation coefficient S20.w, of 18.8 S and a Stokes' radius of 8.5 nm, giving a molecular mass of 679 kDa and a fractional ratio, f/f min, of 1.47. The latter value indicates that the macromolecule is asymmetric or highly hydrated. This large species is composed of four types of polypeptide chains of molecular mass 66 kDa (neuraminidase), 63 kDa (beta-galactosidase), 32 kDa and 20 kDa (carboxypeptidase heterodimer). The 32 kDa and 20 kDa protomers are linked together by a disulphide bridge. Glycopeptidase F digestion of the NGC-complex transformed the diffuse 66-63 kDa band on the SDS gel into two close but sharp bands at 58 and 56 kDa. The two smaller species which were separated on the h.p.l.c. column correspond to tetrameric and dimeric forms of the 66-63 kDa protomers and express exclusively beta-galactosidase activity. Treatment of the NGC-complex with increasing concentrations of guanidinium hydrochloride up to 1.5 M also resulted in dissociation of the complex into the same smaller species mentioned above plus two protomers of molecular mass around 60 and 50 kDa. A model of the largest molecular species as a hexamer of the 66-63 kDa protomers associated to five carboxypeptidase heterodimers (32 kDa and 20 kDa) is proposed